Identification of calmodulin-binding proteins on membranes of secretory granules isolated from bovine neurohypophyses.

Membrane proteins from isolated, purified ox neurohypophyseal secretory granules were separated by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis (PAGE). Using a gel overlay technique, after renaturation procedures, it was demonstrated that 125J calmodulin bound in a Ca2+-dependent way to two protein bands with molecular weights (MW) of 58,000 and 52,000. Binding of small amounts of calmodulin to other protein bands was independent of calcium. No calmodulin binding to granule content proteins could be detected. Treatment of the granules with trypsin prior to separation of membrane proteins removed the Ca2+-dependent binding proteins from the granule membrane. On incubation of granules with [gamma-32P]ATP, protein bands with MW of 52,000 and 45,000 showed a marked phosphorylation activity. The 52,000 band had the same electrophoretic mobility as one of the calmodulin-binding bands. However, no effect of calmodulin on phosphorylation of this band could be demonstrated.